Currently, there are a number of methods to assay or test for male fertility. Current tests test for sperm number, motility, and morphology, for the acrosome reaction, for the release of acrosin, and for the ability to penetrate into zona-free hamster oocytes, as well as the hemizona assay and assays for semen volume and anti-sperm antibodies. While many of these tests provide useful information, there are other mechanisms of sperm fertility failure not diagnosed by these tests, arid no single test is a consistently accurate and reliable test of general sperm fertility. One of the more widely used tests, the penetration of sperm into zona-free hamster oocytes, has proven to be unreliable, providing significant percentages of both false positives (i.e. infertile men who pass the test) and false negatives (i.e. fertile men who fail the test). In addition, no present tests provide an indication of relative potency so as to indicate which sperm, e.g. from among potential donors, has greater fertilizing potential than other sperm.
Semen quality has been analyzed in a number of methods. The first parameter measured is simply volume of the ejaculate (Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction Manual, World Health Organization, 1987). The semen is also screened for ant-sperm antibodies which interfere with fertilization. Anti-sperm antibodies are evaluated in several fashions such as by sperm agglutination, sperm immobilization and immunobead binding. The last example includes some commercially available tests such as SpermCheck (Bio-Rad, Hercules Calif.) and SpermMar (Ortho Diagnostic Systems; Beerse Belgium) and may be used for direct assay (binding to sperm) or for indirect assay (presence of sperm antibodies in the serum) (Khoo, et al., Am. J. Reprod. Immunol. 26:11-16, 1991).
Semen has also been screened for the presence of tumor associated trypsin inhibitor (TATI) (Barnti, et al., Scand. J. Clin. Invest. 51 (Suppl. 207):51-53, 1991). Acrosin, an enzyme important during fertilization, is a serine protease with trypsin specificity. TATI has been shown to be present in semen and shown to be inhibitor of acrosin (Hoppe-Seylers, Z. Physiol. Chem. 365:819-825, 1984).
Sperm are also directly analyzed for a number of criteria. First, the sperm may be examined for normal morphology, total number of sperm, and number of viable sperm using a light microscope (Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction Manual, World Health Organization, 1987).
Secondly, sperm may be examined for motility. Motility has been divided into two types: percentage of motile sperm vs. immotile sperm and percentage of motile sperm with forward progression vs. immotile sperm.
Two other tests of sperm activity are used, the sperm motility index, which is a measurement of disturbances in optical density of the semen (Bartoov, et al. Fertil. Steril. 56:108-112, 1991) and the resazurin reduction test, which measures reduction of the dye resazurin by a color change reaction (Glass, et al. Fertil. Steril. 56:743-746, 1991).
The integrity of sperm plasma membranes are evaluated using the hypo-osmotic swelling test (HOS-test). In this test, sperm are evaluated for percentage showing swelling in hypo-osmotic media (Smith, et al., Int. J. Androl. 15:5-13, 1992). There is debate as to whether there is also significance in differential tail swelling patterns.
A popular test conducted to evaluate sperm quality is the hamster oocyte penetration test (HOP-test), sometimes referred to as the "humster" assay, first referenced above. Sperm are added to media containing zona free hamster oocytes. The hamster oocytes are then evaluated for penetration by the sperm and for decondensation of the sperm nucleus. This assay also analyzes the sperm's ability to capacitate and undergo the acrosome reaction, because these events are necessary for penetration (Yanagamachi, et al., Biol. Reprod. 15:471-476, 1976). While this test is sanctioned by the National Institutes of Health (NIH), and it is used commercially by the medical community, its predictive value is subject to question.
There are a number of industries which also evaluate sperm quality, for example, for the artificial insemination of domestic farm animals and other animals such as dogs, thoroughbreds, and llamas. In the case of domestic farm animals, the primary methods of evaluation are non-return rate, which is the percentage of bred animals (artificial insemination or natural breedin) which do not return into estrus, and the in vitro fertilization rate of homologous oocytes. In those animals which are naturally bred rather than artifirially inseminated, the only evaluation is non-return rate. Therefore, there is a need in the art of reproductive biology for an assay that will evaluate the reproductive quality of a sperm sample.
There is also a need in the art for an assay that will evaluate the reproductive ability of sperm samples and identify and test for other modes of sperm reproductive failure.